The present invention is an improvement to those methods for the detection of peroxidase activity in samples of body fluid whose sensitivity is impaired by hemoglobin interference. For example, there is disclosed in U.S. Pat. No. 5,374,561 a method for the detection of creatinine in urine which comprises contacting a urine sample suspected of containing creatinine with cupric ions, a hydroperoxide and an oxidizable dye which provides a colored response in the presence of oxygen free radicals and a hydroperoxide. In this assay, the first step involves the formation of a Cu.sup.++ .cndot.creatinine chelated complex. The oxidizable dye is oxidized by the transfer of an electron therefrom to the Cu.sup.++ .cndot.creatinine complex to provide the non-reactive Cu.sup.+ .cndot.creatinine form which is regenerated to Cu.sup.++ .cndot.creatinine by the loss of an electron to the hydroperoxide. This assay works well in the absence of hemoglobin, but in the presence of hemoglobin its precision is affected due to the tendency of hemoglobin to oxidize the dye thereby causing false positives. Since hemoglobin is frequently found in body fluids such as urine, there is a need to block its interference to thereby provide a more precise assay.
In co-pending application U.S. Ser. No. 08/990,389; there is disclosed an assay for an analyte in a fluid test sample which involves combining the fluid test sample with a reagent system comprising an apo-peroxidase, a redox dye, a hydroperoxide and a transition metal porpyhrin chelate which is bound to an analyte/analyte specific binding partner having a combined molecular weight of at least about 180 K Daltons. When this conjugate interacts with analyte in the fluid test sample, a portion of the specific binding partner is dissociated from the conjugate thereby enabling the metal porpyhrin to reconstitute with the apo-peroxidase which reconstituted peroxidase can interact with the hydroperoxide and redox dye to provide a colored response to analyte in the fluid test sample. This type of assay is also adversely affected by the presence of hemoglobin in the fluid test sample because of oxidation of the redox dye in the presence of a hydroperoxide.
Other assays involving the use of metal chelates include ferric ion/creatinine; transition metal/2,2'-dipyridyls; transition metal/diethylene polyamino polyacetic acids such as EGTA, HEDTA and NTA; transition metal/oximes; transition metal/phenianthrolines; transition metal/throcarbamides; transition metal/triethylene tetramine and transition metal catechol. The use of iron chelates, iron creatine in particular, to detect hydrogen peroxide is disclosed in U.S. Pat. No. 5,702,955.